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PATHOLOGY ONCOLOGY RESEARCH
Vol 7, No 1, 2001
10.1053.paor.2001.0308 available online at http://www.idealibrary.com on
The Role of Matrix Metalloproteinases in Tumor Angiogenesis and Tumor Metastasis
Anitha JOHN,1 George TUSZYNSKI1,2
Medical College of Pennsylvania and Hahnemann University School of Medicine
Department of Pathology1 and Surgery2, Philadelphia, USA
Although a considerable amount of effort has beenof enzymes that has been shown over the years toplaced on discovering the etiologies of cancer, theplay a role in tumor progression is the matrix met-majority of the basic cancer research existing todayalloproteinase (MMP) family. The main functionhas focused on understanding the molecular mech-of MMPs, also known as matrixins, is degradationanism of tumor formation and metastasis. Metasta-of the extracellular matrix physiologic functiontic spread of tumors continues to be a major obsta-involving MMPs include wound healing, bonecle to successful treatment of malignant tumors.resorption and mammary involution. MMPs, how-Approximately 30% of those patients diagnosedever, also contribute to pathological conditionswith a solid tumor have a clinically detectableincluding rheumatoid arthritis, coronary arterymetastasis and for the remaining 70%, metastasesdisease, and cancer. Tumor cells are believed to uti-are continually being formed throughout the lifelize the matrix degrading capability of theseof the tumor. Even after the tumor is excised, theenzymes to spread to distant sites. In addition,threat of death is attributable to the metastasis thatMMPs also are thought to promote the growth ofmay occur through the remaining tumor cells. Inthese tumor cells once they have metastasized.addition, treating the metastasis often proves futileThis review will discuss the role of MMPs andsince metastasis often vary in size, composition,their inhibitors in tumor invasion, angiogenesisand anatomical location. New treatments blockingand metastasis with special emphasis on thethe formation of metastasis will provide greatergelatinases, MMP-2 and MMP-9. chances of survival for cancer patients. One family
logy Research Vol 7, No 1, 14–23, 2001)
Keywords:
matrix metalloproteinases; metalloproteinase inhibitors; gelatinases; tumor angiogenesis; metastasis
Introduction to the MMP familyReceived:
Febr 1, 2001; accepted: Correspondence: George P. TUSZYNSKI, MCP-Hahnemann
The first mammalian MMP, interstitial collagenase, was
School of Medicine, MS 435, Broad & Vine, Philadelphia, PA19102, USA; Tel: 215 762 6297/6299; fax: 215 762 8787;
discovered over thirty years ago by Gross and Lapiere in
amphibian tissue.10 Since then, over 14 members have
List of Abbreviations
been added to the family. The MMP family can be subdi-
Amino-terminal – NH2-terminal; Carbohydrate Antigen 19-9 –
vided into five groups: the collagenases, the stromelysins,
CA19-9; Carcinoembryonic Antigen – CEA; Cerebrospinal Fluid –
the gelatinases, PUMP-I or matrilysin, and membrane-
CSF; Endothelial Cell – EC; Extracellular Matrix – ECM; Human
type (MT) MMPs. Although the classification system was
Dermal Microvascular Endothelial Cell – HDMVEC; Human
developed on the basis of substrate specificity, it is now
Umbilical Vein Endothelial Cell – HUVEC, Interleukin-1 Alpha –IL-1α; Interleukin-1 Beta – IL-1β; Interleukin-8 – IL-8; Intravital
recognized that there is some overlap between some mem-
Videomicroscopy – IVVM; Matrix Metalloproteinase – MMP;
bers of the family (see
Table 1). For example, MMP-2 has
Matrix Metalloproteinase Inhibitor – MMPI; Membrane-Type Met-
been reported to have the ability to cleave fibrillar colla-
alloproteinase – MT-MMP; Phorbol 12-Myristate 13-Acetate –
gen similar to the collagenases.11
PMA; Polymorphonuclear Cells – PMNs; Thrombospondin-1 –
This multigene family of metal containing proteases
TSP-1; Tissue Inhibitor of Metalloproteinase – TIMP; Transform-ing Growth Factor Beta – TGF-
share several common characteristics: (1) each degrade at
β; Tumor Necrosis Factor – TNF;
Vascular Endothelial Cell Growth Factor – VEGF
least one component of the basement membrane; (2) they
2001 W. B. Saunders & Company Ltd on behalf of the Arányi Lajos Foundation
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Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis
are active at physiological pH; (3) they require 2 Zn++
transcriptional activation is not fully understood. These
ions/molecule in order to be active; (4) they are inhibited
factors cause variable patterns of expression in different
by metal chelators and tissue inhibitors of metallopro-
tissues and have variable effects on the different MMP
teinases (TIMPs); and (5) they are secreted as zymogens
family members, complicating the understanding of gene
and require activation extracellularly.12,13
regulation of MMPs in both physiological and patholog-
Comparing the protein and cDNA sequences of cloned
ical states. One of the most potent family of inducers are
MMP molecules reveal a number of conserved regions
phorbol esters such as PMA.8,15 Other inducers include
within the family. All latent MMPs contain at least three
interleukin (IL)-1α, IL-1β, IL-8, transforming growth
domains: (1) a hydrophobic pre-peptide domain that is nec-
factor (TGF)β-1, and tumor necrosis factor (TNF).8,9,13
essary to signal secretion, (2) an amino terminal propeptide
Other factors implicated in stimulating MMP regulation
domain which is removed upon activation, and (3) the Zn++
include basic fibroblast growth factor (bFGF), epidermal
containing catalytic domain.8,12,13 This basic 3-part struc-
growth factor (EGF), and vascular endothelial cell
ture is present in all of the MMPs. Within the family, how-
growth factor (VEGF).16-18 Important to remember is that
ever, further subdivisions exist based on distinct structural
there is often a balancing effect within the corresponding
variations. Matrilysin (MMP-7), the smallest member of
physiological inhibitors of MMPs, the tissue inhibitor of
the MMP family is comprised of only the basic core struc-
metalloproteinases (TIMP). To create a favorable state
ture. The remaining enzymes all contain a "hemopexin"
for invasion, there must be a balance of protease to
domain connected to the catalytic domainby a hinge region. The hemopexin domain
Table 1. Matrix Metalloproteinase Family*
contains the TIMP binding site and may
Molecular Weight (kDa)
also be involved in receptor binding. The
MMP Name and NoECM Substrate
membrane-type metalloproteases (MT-MMPs) are a unique subdivision in that
Interstitial Collagenase
Fibrillar Collagens
they contain a transmembrane domain at
the carboxyl terminal anchoring the mole-
Neutrophil Collagenase
Fibrillar Collagens
cule to the cell surface. Another subdivi-
Fibrillar Collagens
sion, the gelatinases (MMP-2 and MMP-
9) contain a fibronectin-like domain with-
Fibrillar Collagens
in the catalytic domain responsible forcollagen binding. MMP-9 has an addi-
Laminin, fibronectin,
tional collagen V-like sequence within the
non-fibrillar collagens
Laminin, fibronectin,
catalytic domain downstream of the Zn++
non-fibrilar collagens
Laminin, fibronectin,
non-fibrillar collagens
Laminin, fibronectin,
non-fibrillar collagens
Regulation of the MMPs occurs on
three levels: alteration of gene expres-
sion, activation of latent zymogens, and
Gelatinase A, fibrillar
inhibition by tissue inhibitors of metallo-
proteinases. The cooperative effects of
ECM glycoproteins
these three factors provides a tightly con-
trolled regulation of MMPs in normal
physiological states. Alteration of all
three levels of control have been associat-
Type I, IV, V and fibrillar
ed with tumor cell progression.14
Collagens; gelatin
Type IV, V collagen,
MMPs and TIMPs are thought to be
regulated by a variety of cytokines,
*The MMP family are listed here in addition with their molecular weights and theirsubstrates.
growth factors, steroid hormones and
The subdivisions correspond to a combination of structural and substrate homo-
phorbol esters.8 Despite a vast array of
logy. The newest members including MMP-18, MMP-19, and the MT-MMPs have
research in this area of MMP regulation,
not yet been fully characterized.
Vol 7, No 1, 2001
JOHN and TUSZYNSKI
inhibitor in order for the cell to migrate and invade.
or stromelysin. There seems to be a tighter regulation of
Although the exact role of cytokines and growth factors
MMP-2 than the other MMPs perhaps in part to the fact
is not clear, it appears that they act in conjunction to reg-
that MMP-2 is the most commonly expressed MMP in
ulate both MMPs and their inhibitors to create the envi-
normal tissues.28 The newest subclass of MMPs, the
ronment necessary for either physiological or pathologi-
MT-MMPs, have the capacity to activate pro-MMP-2.29
The transmembrane domain is essential to MT-MMP's
In addition to cytokines and growth factors, MMP pro-
capacity to activate MMP-2. Understanding this level of
duction can be regulated by environmental factors. For
regulation of MMP will provide yet another target for
example, extracellular matrix (ECM) component peptides
development of anti-cancer therapeutics.
have been observed to have an effect on MMP production.The laminin-1 peptide, AG-73 has been shown to enhance
Inhibitors of Metalloproteases
gelatinase production and increase in vivo B16-F10melanoma cell lung and liver metastasis.19 Vitronectin,
The third level of control of MMPs occurs through inhi-
collagen, and elastin have also been shown to induce col-
bition of enzymatic activity. Various physiological agents
lagenases and gelatinases expression in either tumor cells
can have an inhibitory effect on MMPs including
or fibroblasts.20-22 Our laboratory has done extensive work
macroglobulin and TIMPs, tissue inhibitors of metallopro-
in identifying thrombospondin-1 (TSP-1), a 450 kDa
α2-macroglobulin molecule, a large mole-
extracellular matrix protein, as a stimulator of MMP-9
cular weight (780 kDa) serum protein, can inhibit pro-
production in endothelial cells, pancreatic cancer cells,
teinases, but its size prevents the molecule from entering
and breast cancer cells.23,24 The capacity of TSP-1 to up-
into tissue spaces. The TIMPs are much smaller molecules
regulate MMP-9 is mediated in part by the CSVTCG pep-
and are expressed in various tissues and fluids. There are
tide sequence present in the type one properdin repeats of
four members of the mammalian TIMP family, TIMP-1,
TSP-1. Synthetic peptides containing the CSVTCG
TIMP-2, TIMP-3, and TIMP-4.31 The amino-terminal
sequence probably bind and block a specific TSP-1 recep-
domain present in all TIMP molecules is responsible for
tor.24 These peptides represent a potential therapeutic
the MMP inhibitory activity. The TIMPs form high affin-
approach for the treatment of metastasis. These data all
ity, non-covalent complexes with all active MMPs in a 1:1
suggest that the cell environment plays a key role in regu-
stoichiometric ratio. In addition, TIMP-1 and TIMP-2 can
lating MMP production.
block the pro forms of MMP-9 and MMP-2, respectively.The balance between protease and inhibitor is critical in
Activation of Latent MMPs
determining net proteolytic activity.
TIMP-1 is a 28.5 kDa glycoprotein that has a wide vari-
Another level of MMP regulation is the activation of
ety of functions including growth factor activity, stimulat-
the zymogen/proenzyme secreted form of MMPs. All
ing cell morphology changes, and inhibiting angiogene-
MMPs are secreted as zymogens with the exception of
sis.31 Increased TIMP-1 levels have traditionally been
the MT-MMPs, the membrane sequestered subdivision.
associated with reduced invasion and metastasis. Some
These latent zymogens must be activated in order to
controversy has arisen on the role of TIMP-1, however.
degrade matrix components. As mentioned previously,
Recent studies have shown that TIMP-1 may increase the
these proenzymes remain in an inactive form through an
invasive capacity of tumor cells due to its growth factor
interaction between a cysteine in the proregion and the
like activity.32 TIMP-2, similar to TIMP-1, is associated
Zn++ ion in the active site. This interaction blocks access
with decreased metastatic potential. TIMP-2, a non-glyco-
to the active site and cleavage of this site results in
sylated 21 kDa protein, suppresses tumor growth and inva-
enzyme activation. Trypsin 2 has the ability to activate
sion.31 In contrast to TIMP1 and TIMP-2, which are
both free proenzyme and proenzyme:TIMP-1 complexes,
secreted, TIMP-3 is associated with the extracellular
the major form of MMP
in vivo.25 Other agents which
matrix.31 A recent report describes melanoma cells trans-
have been shown to activate MMPs include cathepsins G,
fected with the TIMP-3 gene reduced invasion and
B, and L, PMN elastase, and hypochlorous acid.26 In
induced cell death in these cells
in vitro.33 The most
addition, the plasmin/plasminogen system has been
recently identified molecule is TIMP-4, a 24 kDa protein.
implicated in tumor cell invasion in part through its abil-
Some controversy exists on the roles of TIMP-2 and
ity to activate MMPs.27
TIMP-3 as well since several studies report increased
MMPs, once activated, are also capable of activating
expression of these compounds results in increased
themselves. MMP-3 can activate MMP-2 and MMP-9 as
metastatic potential. Again, it is important to consider the
can MMP-7. The gelatinases, MMP-2 and MMP-9, can
ratio of protease to inhibitor which is critical to invasive
activate each other. In contrast to other members of the
capacity. A small change in the balance could have a pro-
family, MMP-2 does not seem to be activated by plasmin
found effect on proteolysis.
PATHOLOGY ONCOLOGY RESEARCH
Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis
Tumor progression:
These processes are then repeated upon extravasation of
the angiogenic and metastatic cascade
tumor cells from the vessel.38 In vitro and
in vivo studies
show a definite correlation between gelatinase expression
Metastasis, a hallmark of cancer, is the colonization of
and metastasis. In addition, MMPs are essential factors in
distant sites with cells from the primary tumor.3 The
tumor angiogenesis (see
Figure 1).39 Proteolytic activity is
process of metastasis involves the survival of the primary
required during the formation of the capillary bud in order
tumor cell through a series of steps including: tumor cell
for the endothelial cell to migrate out through the peri-cap-
invasion enabling it to break free from the primary tumor,
illary membrane and through the ECM. In addition, cap-
intravasation, survival and extravasation into and from the
illary elongation, lumen formation, and ECM remodeling
blood or lymphatic circulation, and finally tumor cell col-
all require proteolytic activity. Recent studies have impli-
onization and angiogenesis to form the metastatic lesion.3
cated MMPs as an important protease component in angio-
Although the majority of those cells that escape from the
genesis perhaps as a downstream modulator to known
primary tumor will not survive, those capable of invading
angiogenesis-related molecules such as VEGF and throm-
into and out of the circulatory system will each give rise to
a metastatic tumor.
Metastasis can be described clinically as four different
The role of MMPs in tumor angiogenesis
patterns: No metastasis is present at the initial diagnosisbut appears within (1) months or (2) years after the prima-
Many studies have shown that endothelial cells are
ry tumor is removed; (3) metastasis is present at the time
capable of differentially expressing and activating MMPs
of diagnosis of the primary tumor; and (4) only the metas-
and TIMPs. Type I collagen is the predominant constituent
tasis is detectable at the time of diagnosis.34 Metastasis that
of the perivascular ECM, and as mentioned previously, a
appears after several years have been termed "dormant"
variety of MMPs are capable of degrading collagen type I
metastasis. Considerable research has been done to under-
including interstitial collagenase and neutrophil collage-
stand the molecular changes a normal cell undergoes to
nase.39 Several studies have shown that MMP-1 is a criti-
become malignant. However, no single gene has been
cal protease in the angiogenic cascade. Immunofluores-
implicated as a metastasis-specific gene. There is likely to
cent staining revealed that more aggressive skin tumors
be a cascade of events that occur to create a malignant cell.
displayed a higher number of collagenase-containing
Angiogenesis or neovascularization is a tightly regulated
blood vessels.41 This collagen-degrading activity has been
system occurring rarely in a normal physiologic environ-
shown to be specific through a number of specific MMP
ment.34 Reproduction, embryogenesis, and wound healing
inhibitors such as TIMP, Batimastat, and Marimastat
are among the few situations requiring angiogenesis. The
which are capable of suppressing MMPs and therefore
formation of these new blood vessels is one of the major
capillary tube formation
in vivo.42
factors that stimulates tumor growth. Tumors without a
A number of angiogenic factors have been shown to reg-
blood supply are only able to grow 1-2 mm3, and formation
ulate the production of MMPs in endothelial cells. Our
of a blood supply is critical to forming metastases.35 Atumor cell cannot escape from the primary tumor until thetumor has been vascularized and once the metastatic cell
has reached its target organ, angiogenesis is required forthe metastases to grow.34 Angiogenesis occurs through a
series of steps including (1) the release of angiogenic fac-
tors (2) the release of proteolytic enzymes to degrade thebasement membrane of the postcapillary venule (3) EC
migration toward the tumor (4) EC proliferation and (5)microvessel formation and differentiation.36 Tumors which
bear a higher percentage of angiogenic cells have been
shown to have increased metastatic potential and are more
aggressive tumors.37 Similarly, agents which are capable ofinhibiting angiogenesis, such as anti-bFGF, subsequently
reduce growth of malignant cell lines
in vivo.37
Figure 1. Schematic of tumor growth dependent on angiogene-
Proteases are required by the malignant cell to invade
sis. Tumor cells multiply and grow, eventually requiring a
the ECM. The metastatic cell uses proteases to invade
blood supply to sustain further growth. Cytokines secreted by
through the basement membrane and its underlying con-
the tumor cells and adjacent stromal elements stimulate
nective tissue and then subsequently through the basement
endothelial cells to produce MMPs needed for endothelial cell
membrane of the small blood vessels and lymphatics.migration and tube formation.
Vol 7, No 1, 2001
JOHN and TUSZYNSKI
laboratory has show that TSP-1 can also stimulate BAECs
tor mediated through the lack of MMP-9 expression.
to secrete MMP-9.24 The role of TSP-1 in angiogenesis
These data suggest that MMP-9 plays a regulatory role in
has been controversial. We have shown that TSP-1 is a
angiogenesis not only through proteolytic activity but also
bifunctional modulator of angiogenesis acting in part
through other downstream angiogenic factors.46
through MMP-9 regulation. In a collagen gel tube forma-tion assay, TSP-1 stimulated endothelial tube formation at
Evidence for MMPs in tumor metastasis
low concentration (5
µg/ml) while at higher concentra-
µg/ml), TSP-1 inhibited tube formation. The
In order for a tumor cell to intravasate and extravasate,
ability of TSP-1 to either induce or inhibit tube formation
the collagen-rich ECM and basement membrane must be
was attributable to the amount of MMP-9 produced. Opti-
degraded. This degradative ability can be through either
mal MMP-9 levels were induced by lower concentrations
enzymatic capacities of the tumor cell or through enzy-
of TSP-1. Increasing amounts of MMP-9 caused excessive
matic activity of cellular components of the matrix, such
proteolysis and inhibited tube formation.
as fibroblasts. Likely there is a cooperation between the
The roles of the type IV collagenases, MMP-2 and
two components enabling the tumor cell to reach its target
MMP-9, in angiogenesis have been explored in a variety
organ and survive. Although all five major classes (serine,
of studies. In a tube formation assay model, MMP-2 was
aspartic, cysteine, threonine, and metalloproteinases) are
produced by HUVECs as they formed tube like structures
involved in metastasis, a great deal of emphasis has been
and this formation could be inhibited by adding TIMP-1 or
placed on the type IV collagenases, MMP-2 and MMP-9.38
TIMP-2 to the cultures.43 A recent study examining the
Type IV collagen is a major structural protein in the base-
progression of mycosis fungoides, a hematological tumor
ment membrane and ECM. A number of studies have
of T-cell lineage, revealed that the more advanced the
linked elevated MMP-2 and MMP-9 levels with an
tumor, the microvessel density increases as does MMP-2
increased metastasis. The conclusions which can be drawn
and MMP-9 expression.44 The MMP-2 was expressed
thus far are that the number and the relative levels of
mostly by the microvascular cells of the blood vessels
MMPs increase with tumor progression.
within and surrounding the tumor in addition to fibroblasts
Several recent studies have been done to try and charac-
adjacent to the tumor stroma. MMP-9 was present in the
terize the phenotypic and enzymatic profiles of more
tissue macrophages located close to the tumor nodules. In
aggressive tumor cell lines. Selection of progressively
contrast, both MMP-2 and MMP-9 were only weakly
more invasive human lung carcinoma cells from an estab-
expressed in normal tissue.
lished CL1cell line, revealed that the more invasive cells
Perhaps the greatest evidence associating MMP-2 in the
had a higher expression of MMP-9.47 These cells had a 4
angiogenic process is the recent MMP-2 knock-out model
to 6 fold increase in invasive activity over the parentals
examining angiogenesis and tumor progression.45 Gelati-
and had an increased metastatic potential in vivo. MMP-9
nase A deficient mice displayed reduced tumor induced
has also been shown to be overexpressed in advanced
angiogenesis as measured by the dorsal air sac assay. In
stage melanoma cells.48 Cell lines from early stage
addition tumor volumes of B16-BL6 melanoma cells and
melanoma lesions revealed no MMP-9 expression while
Lewis lung cancer cells when injected intradermally
those cells from advanced lesions not only expressed
decreased by 39% and 24%, respectively. The number of
MMP-9 but were also capable of being induced to secrete
lung colonies also decreased by 54% for the B16-BL6
more MMP-9 by TGF, IL-1 and TPA.48 Other tumor mod-
melanoma cells and 77% for the Lewis lung cancer. There-
els involving MMP-9 in their invasive phenotype include
fore, host derived gelatinase A is necessary to promote
human non-hodgkins lymphoma cells and human giant
tumor angiogenesis and tumor progression.
cell tumors.49,50 Examining MMP-9 production in human
Similarly, the MMP-9 knock-out model also provides
giant cell tumors, tumors characterized by frequent vascu-
key evidence to the role of gelatinase B in angiogenesis.
lar invasion, revealed that MMP-9 is highly expressed by
MMP-9 knock-out mice exhibit an abnormal pattern of
the giant cells and the absence of collagen and laminin in
skeletal growth plate vascularization and ossification. This
the basement membrane correlated with those regions of
aberrant pattern of vascularization returns to normal after
high MMP-9 expression. MMP-9 through all of these data
transplanting wild-type bone marrow cells indicating that
is certainly one of the key enzymes involved in invasion
MMP-9 expression in cells of bone marrow origin is one
and metastasis.
factor that regulates normal vascularization in the skeletal
MMP-2 has also been observed to be overexpressed in
growth plate. Important to note, however, is that vascular-
more aggressive tumor cells. Comparing a highly metasta-
ization, apoptosis, and ossification compensates to pro-
tic mouse mammary tumor cell line to its parental poorly
duce a normal growth plate after three weeks postnatal in
metastatic cell line, show the highly metastatic cells
these animals. The control on angiogenesis is believed to
express more MMP-2.51 The level of pro-MMP-2 vs.
be mediated by a delayed release of an angiogenic activa-
active MMP-2 also plays a role in determining invasive
PATHOLOGY ONCOLOGY RESEARCH
Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis
and metastatic capacity. The MMP-2 activation ratio in
localization of the MMPs in tumor tissue is similar to that
tumor tissue was also higher in pancreatic carcinoma
of normal tissue. Although there are several studies show-
patients with positive regional lymph nodes than those
ing MMP-2 and MMP-9 production to be localized to the
without metastasis.52 The increase in MMP-2 activity lev-
tumor cells, the majority show MMP-2 and MMP-9 to be
els correlates with the metastatic potential of the two car-
produced by the tumor stromal elements. Immunohisto-
cinoma types. Elevated levels of MT-MMPs have been
chemical studies of several types of carcinomas including
shown to increase activated levels of MMP-2 in breast,
ovarian, thyroid, hepatocellular, and gastrointestinal show
cervical, and lung carcinomas producing higher levels of
MMP expression in stromal fibroblasts.56-59 In both ovari-
invasiveness and metastasis. Transfection of several cell
an and hepatocellular cancers, MMP-2 was localized to
lines (HT-1080 fibrosarcoma, MCF-7 breast carcinoma,
the tumor cell membrane at both the invasive fronts and at
and U251.3 glioma cell lines) with the MT-MMP gene
sites of vascular invasion.58,59 In breast carcinoma, tumor
caused an increase in tumor cell invasion and migration.53
cells themselves were capable of modulating MMP-2
Cell lines displaying an intermediate level of activation
binding via MT1-MMPs as well as via TIMP-2 and Vβ3
were the most invasive while those cells with a high level
integrins.60 An explanation as to why both the tumor cells
of activation were the least invasive. This is probably due
and the adjacent stromal cells express MT1-MMP, MMP-2,
to the balance required between MMPs and TIMP to cre-
and MMP-9 may be that both cellular components con-
ate a controlled proteolytic system.
tribute to a different part of the metastatic cascade. The
Although the major role of MMPs in metastasis has
tumor cell MMPs may contribute to the invasive growth of
been inferred from the
in vivo and
in vitro data presented
the tumor while the stromal elements contribute to the
above to be breakdown of the ECM, recent studies have
remodeling process and the desmoplastic reaction that
proposed additional roles for the MMP family. A recent
occurs in the tissue adjacent to the tumor.
review summarized these new roles for MMPs in invasionand metastasis.38 Most of the
in vivo and
in vitro assays
MMPs and clinical therapeutics
designed to examine the role of MMPs on tumor invasion
MMPs as diagnostic markers
measure the end results, as in the number of micrometas-tases formed. The mechanism, however, remains
Numerous studies have shown the higher the MMP
unknown. Intravital videomicroscopy (IVVM) allows for
expression in the tumor, the more aggressive the cancer.
the observation of the metastatic cascade by following the
Measuring the MMP level in the serum, plasma, or CSF has
tumor cell through the microcirculation. The results fromthese experiments suggest that the destruction of tumorcells in the circulation and during extravasation do not
contribute as much as previously thought to the inefficien-cy of metastasis. Rather, the growth of the individualtumor cell once in the target organ appears to be the rate
limiting step.38 Tumor cells engineered to overexpress
TIMP-1 were shown to extravasate at rates equal to wild-
Angiogenic Stimuli
type cells but were unable to form proliferative colonies
within the target organ.54 Although these data suggest that
MMPs may play a role in tumor cell growth, the studies ofMMPs involvement in ECM degradation and basementmembrane invasion still support the core role of MMPs inmetastatic invasion. The steps in which MMPs play a roleare summarized in
MMP expression in various tissue types has been exam-
Figure 2. Roles of MMPs in tumor cell metastasis. The roles ofMMPs in metastasis are multifold. The steps where MMPs are
ined through immunohistochemical analysis and in situ
involved are depicted in this schematic. 1) MMPs are involved
hybridization. MMPs are expressed in a variety of tissues
in primary tumor growth and angiogenesis; 2) MMPs are
and are a necessary component of the normal tissue
secreted by both tumor cells and stromal cells upon cytokine
remodeling process that occurs during wound healing,
stimuli thus enabling the tumor cell to invade and intravasate;
pregnancy, and bone resorption. MMP-2 seems to be the
3) Extravasation of the tumor cell; 4) Tumor cell migration in
most common enzyme expressed in normal tissues, with
ECM of distant site; 5) Growth and angiogenesis of distant
expression being the highest in stromal elements.55 The
Vol 7, No 1, 2001
JOHN and TUSZYNSKI
received some attention as a possible pre-
Table 2. Metalloproteinase Inhibitors and Their Clinical Uses*
dictor of tumor stage, metastasis, and
recurrence. A study examining the serum
Class NameClinical Use
levels of MMP-2 and MMP-9 in gastriccarcinoma patients revealed a higher level
of both pro-enzymes in the cancer patients
TIMP-1 (28.5 kDa) Inhibits all activated MMPs;
Possible target for
vs. healthy volunteers.61 In addition, the
inhibits pro-MMP9
gene therapy; basis
MMP level may be used in conjunction
for synthetic MMPI development
with other markers, such as CEA for colon
Inhibits all activated MMPs;
carcinoma, to better determine the stage of
inhibits pro-MMP2
the tumor. Other studies show that there isa correlation with MMP levels and tumor
metastasis. Similar results were observed
Substrate analog to all
Used to generate
in prostate cancer where serum MMP-2
preclinical data formarimastat
levels were higher in the carcinomapatients vs. healthy and benign prostatic
Substrate analog to all
Used in clinical tri-
hyperplasia patients, and among the can-
als in colon, pancre-
cer cases, there was a higher level in those
atic, ovarian cancer,
patients with metastasis.62 In addition to
correlating with metastasis, MMP levels
Weak MMP inhibitor
Used in rheumatoid
can also predict the recurrence of tumors.
arthritis and peri-
In a study examining the recurrence of
urothelial cancer after resection showed
(clinical Trials)
that those patients with recurrence had a
Chelates zinc, inhibiting
In vitro data sug-
higher MMP-2:TIMP-2 serum level than
gests use in malig-
those without recurrences.62 Also, an inde-
pendent study showed a higher MMP-2/TIMP-2/MT1-MMP level in bladder
* A sample of MMPIs are listed here along with their potential clinical uses. MMPIsare still in clinical trials. They constitute a new class of emerging drugs targeting
cancer patients is associated with
angiogenesis and metastasis formation.
decreased survival.63
Metalloproteinase inhibitors: chemotherapeutic agents
melanoma cells, and a human astrocytoma cell line trans-fected with the TIMP-1 gene showed metastasis with a
As explained previously, the TIMPs are one mechanism
reduced growth when injected into nude mice.65-67 Addi-
of control used physiologically to regulate MMP function.
tional evidence includes the ability of monoclonal anti-
A number of studies have shown that overexpression of
bodies against gelatinases to inhibit invasion in
TIMPs produce a less metastatic phenotype presumably by
assays.68,69 These data all suggest a role for MMPIs as a
reducing the amount of active metalloproteinases.
possible anti-cancer therapy. By altering the MMP-TIMP
Although the initial evidence for use as possible chemo-
ratio, the invasive and metastatic ability of tumor cells can
therapeutic agents came from the studies involving natur-
al TIMPs, the inability to mass produce and orally formu-late natural TIMP cost-effectively have led to the develop-
Synthetic MMPIs
ment of synthetic MMPs. Some more common MMPIs arelisted in
Two of the prototype MMPIs today are batimastat and
marimastat. Batimastat, one of the first synthetic MMPIs,
Evidence for MMPIs as effective anti-cancer agents
has a potent activity against most MMPs with an IC50 inthe low nanomolar range. Batimastat acts by competitive,
Studies with the TIMP molecules provide the basis for
reversible inhibition by mimicing the substrate of MMPs.
developing synthetic MMPIs as anticancer agents. Exper-
The long half-life and the route of administration of bati-
iments with recombinant TIMP-1 have shown that rTIMP-
mastat is convenient for animal models. Marimastat is
1 inhibits the invasion of tumor cells through amniotic
another MMPI with similar inhibitory functions as bati-
membranes.64 Administering rTIMP-1 to mice injected
mastat. Marimastat is almost completely absorbed after
with metastatic B16 melanoma cells also inhibits the for-
oral administration and has a high bioavailability. The half
mation of lung metastasis.64 Gastric cancer cells, B16F10
life of marimastat is approximately 15 hours providing a
PATHOLOGY ONCOLOGY RESEARCH
Matrix Metalloproteinases in Tumor Angiogenesis and Metastasis
convenient twice a day dosing schedule. Marimastat is
bospondin-1 to up-regulate MMP production in both
metabolized quickly in rodents, therefore, it has been used
endothelial cells and tumor cells.24 Thrombospondin-1, a
in clinical trials while batimastat has been used to generate
450 kDa protein, has multiple domains with a variety of
preclinical data.
functions. TSP-1 has been shown to promote invasion in
Preclinical data seem to strongly support the ability of
vitro and in vivo. One of the mechanisms involved is
MMPIs to reduce invasion and spontaneous metastases.
through the up-regulation of MMP-9. Specifically, the
Studies with the B16 murine melanoma model show a
type-1 repeat peptide of TSP-1 has been shown to block
decrease in growth of subcutaneously implanted tumors
the production of MMP-9 in endothelial cells by 86%. We
and reduced spontaneous metastasis formation after surgi-
interpret this result to mean that the peptide alone binds
cal removal of the tumor. The number of lung colonies
and blocks the receptor through which TSP-1 acts. The
formed after IV injection of cells was also decreased by
type I repeat peptides have been shown in mice to block
68%.70 More recently, in another colon cancer model, bati-
the formation of B16 melanoma colonies in the lung. Pep-
mastat treated animals had decreased peritoneal carcino-
tides of matrix components such as the type I repeat of
matosis development and liver metastasis development.
TSP-1 are possible anti-invasive therapies for the treat-
The treated animals also had significantly prolonged sur-
ment of metastases formation. Other MMP inhibitors
vival.71 In addition, a hemangioma model using virus
being studied in clinic trials include AG3340, COL-3,
transformed endothelial cells measured the antiangiogenic
Neovastat, and BMS-275291.
ability of MMPIs through measuring new vessel forma-tion. Batimastat treatment resulted in a decrease in vessel
formation as assessed through hemoglobin content.42
Clinical trials examining the efficacy of marimastat also
The MMPs are a key family of enzymes used by tumor
yielded favorable results. Clinical trials involving MMPIs
cells to invade and metastasize. The up-regulation of these
must involve different parameters than just cytotoxic
enzymes during the invasive state can be caused by a vari-
responses alone because MMPIs are not toxic to the tumor
ety of factors including increased production through
cell. The measure of effectiveness is the actual reduction
cytokines and growth factors, increased activation through
of tumor growth along with the measure of survival time.
mechanisms such as uPa, and decreased inhibition by
The studies involving batimastat included the treatment of
reduced levels of TIMP-1. Developing orally active syn-
malignant ascites and malignant pleural effusion. These
thetic inhibitors of MMPs is a possible treatment for con-
studies seemed to show favorable results; however, the
trolling the metastatic potential of many tumors. While
poor bioavailability and the need to inject the drug direct-
this therapy alone will not destroy the tumor, used in com-
ly into a body cavity led to the clinical trials with marima-
bination with other therapies, these MMPIs could halt the
stat. In a recent review by Steward, an overview of the tri-
disease progression and slow the spread of the tumor.
als with marimastat are provided. Phase I studies revealed
Research still continues in this field and exciting new ther-
the severe joint and muscle pain that occurred with Mari-
apeutic opportunities will ultimately emerge.
mastat at doses of 50 mg twice daily. Symptoms weredecreased by decreasing the dose to 10 mg twice daily.72
Phase II studies have shown a decrease in tumor specif-
ic antigens, a measure of tumor activity, after treatment
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Our laboratory has discovered the ability of throm-
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Vol 7, No 1, 2001
JOHN and TUSZYNSKI
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Vol 7, No 1, 2001

The 4P Tool is designed to assist you in gaining a better insight into your personal Parkinson patient population. For general information or help: mail to [email protected] Prof. A. Jeanjean (Cliniques Universitaires Saint Luc Bruxelles) and Dr. A. Flamez (UZ Brussel, Vrije Universiteit Brussel) are acknowledged for their profound expert advise on the content of this program. The 4P Tool is provided for your convenience, without any warranties, representations or guarantees of any kind.